Recently much research effort has been directed to development of new procedures for introducing nucleic acid sequences into cells. One particular area of focus has been the transformation of cells forming tissues of man and other vertebrate host species to alter the phenotype of the targeted cells. For example, transformation procedures can be used to produce cells that express a biofunctional protein not endogenous to the cell or they can be used to produce cells that express elevated levels of an endogenous, but host deficient, protein. Current methods of introducing exogenous nucleic acid sequences into host tissues requires the harvesting of target cells from the host, transforming harvest cells in vitro with exogenous nucleic acid sequences, and reimplanting the transformed cells into the host.
In accordance with one embodiment of the present invention there is provided a method for introducing nucleic acid sequences into eukaryotic cells in vivo. The method comprises implanting or injecting a novel transformation composition into a host to contact tissue comprising the targeted host cells. The transformation composition comprises intestinal submucosal tissue and a nucleic acid sequence to be introduced into the targeted cell types. Compositions comprising the tunica submucosa and basilar portions of the tunica mucosa of the intestine of warm-blooded vertebrates and their use as tissue graft materials in sheet and tubular form is described in U.S. Pat. Nos. 4,902,508 and 5,281,422, which patents are expressly incorporated herein by reference. The tissue graft compositions described in those patents are used inter alia for vascular graft constructs and tendon and ligament replacement applications. Fluidized forms of intestinal submucosa are described in U.S. Pat. No. 5,275,826 issued Jan. 4, 1994, expressly incorporated herein by reference. Graft compositions comprising intestinal submucosal tissues serve as a matrix for, and apparently help to induce the regrowth of tissues replaced by or in contact with the graft constructs. The present invention is based on the discovery that delivery of exogenous nucleic acid sequences to a focal region of cellular proliferation and regeneration associated with injection or implantation of intestinal submucosal tissue, results in the production of cells containing the nucleic acid sequence and expression of proteins encoded by the nucleic acid sequence. Thus, in accordance with this invention intestinal submucosal tissue, preferably that comprising tunica submucosa and basilar portions of the tunica mucosa delaminated from adjacent tissues of vertebrate intestine, is used as an effective delivery system to introduce exogenous nucleic acid sequences into eukaryotic cells.
The terms “transformed cells” and “transformed tissues” as used herein refers to cells or groups of cells that retain their normal cell cycle, but have new or enhanced phenotypical properties deriving from the presence or expression of exogenous nucleic acid sequences introduced into the cell. The term “exogenous nucleic acid sequences” as used herein refers to any nucleic acid sequence having an origin external to the targeted cells, including recombinant nucleic acid sequences expressed in the targeted cells and/or genes not typically expressed in said cells. Genes that are capable of modifying or altering the phenotype of a cell upon introduction into the cell typically encode proteins functional in cell tissue maintenance and growth, and are generally termed herein as “biofunctional proteins”.
Most present procedures for transforming eukaryotic cells rely upon indirect methods: target cells are removed from the body, infected with viral vectors carrying the new genetic information, and then reimplanted. A direct means of transforming eukaryotic cells (in vivo transformation), is preferred, but not feasible under current viral transformation procedures. Currently, retroviral vectors are the preferred vehicle for introducing DNA into eukaryotic cells. Retroviral vectors provide a high efficiency of gene transfer into replicating cells. However, the preparation of retroviral vectors requires extensive testing to ensure that no replication-competent retroviruses contaminate the vector preparation. Such extensive testing increases the cost of cell transformation procedures. In addition, even after extensive purification of retroviral vectors, the use of these vectors for human applications is still held suspect due to the association between retroviruses and cancer. An additional shortcoming of current retroviral transformation techniques is the inability to directly introduce genetic material into eukaryotic cells in vivo.
One embodiment of the present invention provides in vivo transformation of cells in a host, and thus does not require the removal and reimplantation of host tissue. Such is accomplished by use of a transformation composition including a nucleic acid sequence encoding a biofunctional protein and intestinal submucosal tissue preferably comprising the tunica submucosa, delaminated from the tunica muscularis and at least luminal portions of the tunica mucosa of vertebrate intestine. Upon implantation or injection such compositions are effective for transforming host cells and/or inducing the production of tissue comprising host cells containing and expressing exogenous nucleic acid sequences. Cells/tissues which can be targeted for transformation in accordance with this invention include musco-skeletal tissues and specifically, cells participating in the regeneration and repair of tendon, ligament, cartilage, bone and other connective tissues.
Thus, one aspect of the present invention is a composition useful for the transformation of eukaryotic cells, the composition comprising nucleic acid sequences, preferably recombinant DNA, and submucosal tissue in solid (e.g., sheet or strips) or fluidized form.
In another embodiment of this invention there is provided a method for producing transformed eukaryotic cells by contacting target cells with a transformation composition under conditions conducive to proliferation of the target cells.
Still another embodiment of the present invention is a method for inducing the formation of endogenous tissues comprising transformed cells by implanting or injecting a transformation composition, or its respective components independently, to contact tissue containing target cells in vivo.
Additional objects, features and advantage of the invention will become apparent to those skilled in the art upon consideration of the preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.